Design, preparation, and properties of antibacterial β-peptides

ABSTRACT

An antibacterial β-peptide having the following formula:                    
     wherein 
     R 1  is H, an alkyl group including 1-4 carbon atoms, phenyl, heteroaryl, or an alkyl-aryl; 
     R 2  is an amine-containing alkyl group having the formula —(CH 2 ) m NH 2 , wherein m=1, 2, 3, 4, or 5, (CH 2 ) x NHC═NHNH 2  wherein x is 1, 2, 3, 4, or 5, a pyridyl, an alkylpryidyl, an amidine-substituted benzyl, a phenyl group, or a cyclic amidine; 
     R 3  is H, an alkyl group including 1-4 carbon atoms, phenyl, heteroaryl, or an alkyl-aryl; 
     X is —NH 2 , —OH, —NHR, or OR where R is alkyl, aryl or acyl groups either free or polymer-supported, a carboxamide, a substituted carboxamide, or a polymer; 
     Y is H, an alkyl group, an acyl group, an acyl-terminated polymer, a sulphonamide, an ether, a urea, a urethane, or a polymer; and 
     n is 2, 3, 4, 5, 6, or 7.

This application claims priority to provisional application No.60/170,110 of the same title filed Dec. 10, 1999, the entire contents ofthe disclosure of which is hereby incorporated by reference.

This work was supported by grants from the MRSEC program of the NSF, andNSF grants 9634646 and 9905566.

FIELD OF THE INVENTION

The invention relates to β-peptides, or peptides including P-aminoacids, particularly β-peptides that exhibit antibacterial properties.The present invention also relates to materials that incorporate thepeptides, thereby providing the materials with antibacterial properties.

BACKGROUND OF THE INVENTION

Most proteins typically found in nature are made up of α-amino acids. Ina-amino acids, the amino group is attached to the molecule at theα-carbon atom. The amino group may also be attached to other carbonatoms. For example, in β-amino acids and gamma-amino acids, the aminogroup may be attached to other carbon atoms.

Incorporation of different amino acid forms can result in differences inthe structure of proteins that incorporate the amino acids. In a proteinthat incorporates β-amino acids, the amino group of the amino acid isstill bonded to the carboxylic acid group of an adjacent amino acid,forming an amide bond. When β-amino acids are joined to form a protein,an extra carbon atom will be present in the chain of carbon atoms formedin the protein.

Proteins can have primary, secondary, tertiary and quaternarystructures. The function of a protein may be related to its structuresince the structure may permit the protein to interact with othermolecules or structures. For example, having a certain sequence of aminoacids that fold in a certain manner, a protein may have a complementarystructure to another molecule or portion of a cell structure, therebypermitting the protein to interact with other molecules or cells.Changing the arrangement of the atoms in the amino acids, as withβ-amino acids, and, subsequently, changing a protein or peptide does notalter the nature of the atoms and, for example, charges associated withthe atoms.

SUMMARY OF THE INVENTION

The present invention provides particular β-peptides that haveantibacterial properties. The antibacterial β-peptide can have thefollowing formula:

wherein

R₁ is H, an alkyl group including 1-4 carbon atoms, phenyl, heteroaryl,or an alkyl-aryl;

R₂ is an amine-containing alkyl group having the formula —(CH₂)_(m)NH₂,wherein m=1, 2, 3, 4, or 5, (CH₂)_(x)NHC═NHNH₂ wherein x is 1, 2, 3, 4,or 5, a pyridyl, an alkylpryidyl, an amidine-substituted benzyl, aphenyl group, or a cyclic amidine;

R₃ is H, an alkyl group including 1-4 carbon atoms, phenyl, heteroaryl,or an alkyl-aryl;

X is —NH₂, —OH, —NHR, or OR where R is alkyl, aryl or acyl groups eitherfree or polymer-supported, a carboxamide, a substituted carboxamide, ora polymer;

Y is H, an alkyl group, an acyl group, an acyl-terminated polymer, asulphonamide, an ether, a urea, a urethane, or a polymer; and

n is 2, 3, 4, 5, 6, or 7.

Additionally, the present invention provides antibacterial P-peptideshaving the following formula:

Y—(T)_(n)—X

wherein

n is 2, 3, 4, 5, 6, or 7;

X is —NH₂, —OH, —NHR, or OR where R is alkyl, aryl or acyl groups eitherfree or polymer-supported, a carboxamide, a substituted carboxamide, ora polymer;

Y is H, an alkyl group, an acyl group, an acyl-terminated polymer, asulphonamide, an ether, a urea, a urethane, or a polymer; and

T is a triplet including T1-T2-T3, wherein T1 comprises a hydrophobicβ-amino acid, T2 comprises a polar or basic amino acid, T3 compriseseither a hydrophobic β-amino acid or a polar or basic β-amino acid,wherein at least one-half of the triplets include a basic β-amino acid.

Significanly, the present invention concerns a number of applications ofthese β-peptides to provide antibacterial properties when incorporatedinto other materials.

Still other objects and advantages of the present invention will becomereadily apparent by those skilled in the art from a review of thefollowing detailed description. The detailed description shows anddescribes preferred embodiments of the invention, simply by way ofillustration of the best mode contemplated of carrying out the presentinvention. As will be realized, the invention is capable of other anddifferent embodiments and its several details are capable ofmodifications in various obvious respects, without departing from theinvention. Accordingly, the drawings and description are illustrative innature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

Objects and advantages of the present invention will be more clearlyunderstood when considered in conjunction with the accompanyingdrawings, in which:

FIG. 1 represents a graph that illustrates results of circular dichroismspectroscopy studies on compounds 1 and 2 of group II of compoundsaccording to the present invention;

FIG. 2 represents a graph that illustrates results of membrane bindingstudies carried out on compounds 1 and 2 of group II of compoundsaccording to the present invention;

FIGS. 3a and 3 b represent graphs that illustrate relationships betweenpercent leakage and time demonstrating results of tests of peptideinduced leakage of liposome contents carried out with compounds 1 and 2of group II of compounds according to the present invention;

FIG. 4 represents a graph that illustrates relationships between percentleakage and peptide concentration and fraction bound and peptideconcentration demonstrating results of tests of peptide induced leakageof liposome contents carried out with compounds 1 and 2 of group II ofcompounds according to the present invention; and

FIGS. 5 and 6 represent graphs that illustrate results of tests of theantibacterial properties of peptides according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Many vertebrates and invertebrates secrete natural substances thatpossess both antibacterial and/or indiscriminate cytotoxic properties.Examples of some of these substances include PGLa (frog skin), defensins(human phagocytes), cecropins (Silkmoth pupae or pig intestine),apidaecins (honeybee lymph), melittin (bee venom), bombinin (toad skin)and the magainins (frog skin). Purification of the active constituentsof these natural substances have shown that they consist primarily ofprotein and it has been suggested that they may constitute a system ofcellular immunity in the producing organism.

Peptides and oligopeptides that have activity against microorganismsspan a broad range of molecular weights, secondary conformations andsites of action. Biological activity can range from being specificallybactericidal or fungicidal to being indiscriminately cytotoxic (celllytic) to all cells. Peptides that are specifically bactericidal includelarge polypeptides such as lysozyme (MW 15000 daltons) and attacins (MW20-23,000 daltons) as well as smaller polypeptides such as cecropin (MW4000 daltons) and the magainins (MW 2500 daltons). The spectrum ofbiocidal activity of these peptides is somewhat correlated to size. Ingeneral, the large polypeptides are active against limited types andspecies of microorganisms (e.g., lysozyme against only gram positivebacteria), whereas many of the smaller oligopeptides demonstrate a broadspectrum of antibacterial activity, killing many species of both grampositive and gram negative bacteria.

In part, the antibacterial properties of the P-peptides may stem fromthe three dimensional structures that they adopt. Along these lines, ithas been demonstrated that relatively short (compared with α-peptides)oligomer sequences of β-peptides derived from β-amino acids adoptwell-defined helical secondary structures in both organic and aqueoussolution. It should be possible to design polymers that fold not onlyinto predetermined secondary structures, but also tertiary andquaternary structures. Unlike natural α-peptides, β-peptides have beenshown to be chemically stable and resistant to enzymatic degradation,such as resistance to proteases. As a result, they seem to provide anattractive medium for the construction of biomimetic polymers.

β-peptides have been demonstrated to have remarkable properties.β-peptides can adopt secondary structures such as helices, sheets andturns, which are very similar to the counterparts of natural proteins.The substitution pattern of β-amino acids is found to be an importantdetermining factor of the secondary structure that their oligomers form.Since β-amino acids include two backbone carbon atoms that can bear sidegroups, β-peptides generally have larger diversity of secondarystructures than natural peptides.

The natural antimicrobial peptides that have been isolated from defensesystems of insects, amphibians and mammals have exhibited activityagainst large varieties of bacteria, fungi, and even tumor cells. Anumber of studies have been carried out on their structure activityrelationship, especially the structural class of amphipathic linearpeptides. A number of studies have been carried out on their structureactivity relation (SAR), especially the family of amphipathic linearpeptides. Usually peptides of this type have multiple positive chargesfrom residues such as lysine and arginine. In aqueous solution, thesepeptides often adopt a random coil structure but they form amphiphilicβ-helical structures when they interact with phospholipid bilayers ofcellular membranes. It is believed that this interaction results in adisruption of the cell membrane causes the cell death. The factors thatinfluence their selective antibacterial activities includehydrophobicity, charge distribution, helical propensity and length ofthe peptide. Generally, the more hydrophobic the peptide, the lessselective it is. For peptides that have certain hydrophobicity andcharge distribution patterns, optimum selectivity can be reached at acritical length.

These discoveries in part led to the present invention. Particularlyrelevant were the discoveries related to the folding of the proteins. Atleast in part, a periodicity to hydrophobic residues included in theproteins seems to be particularly important to their folding andsubsequent antibacterial properties.

In general, peptides according to the present invention includemultiples of groups of three β-amino acids. Typically, the peptidesinclude at least two multiples of the groups of three β-amino acids, oralso referred to as triads or triplets herein. As many as 7 multiplesmay be included. At least as many multiples as are necessary to make oneturn of a helix structure typically are included.

It is not necessary that each triplet be the same. Each triplet ofβ-amino acids may be different. One factor that may be significant indetermining which β-amino acids to include in each group is the natureof the groups. In other words, the hydrophobicity, hydrophilicity,polarity, or other characteristics may be important. This is especiallytrue in considering how the peptide will fold in the final molecule andwhether the desired arrangement will result from the selected aminoacids. Typically, the peptides of the present invention form a L+2helix. As discussed herein, molecules may be constructed to havestructures that take on this helical configuration in relevantconditions.

In one embodiment, each triplet of β-amino acids includes a hydrophobicβ-amino acid on each end with a β-amino acid having a polar side chainin the middle. However, as stated above, each group of three amino acidsneed not be the same. In some cases, it is sufficient for at leastone-half of the β-amino acids to be a basic β-amino acid.

Typically, the β-amino acids that are utilized in the present inventioninclude alanine, valine, leucine, and lysine. However, substitutions maybe made on these amino acids, at any carbon atom, but particularly atthe second and third carbon atoms, the carbon atoms adjacent where thecarboxylic acid group is present and the next carbon atom down themolecule.

A number of groups may be attached to the ends of the peptide. Alongthese lines, the peptide may terminate in the carboxylic acid and aminegroups. However, more typically, additional groups may be attached tothe peptide. A number of groups are listed below. However, a significantaspect of the present invention is that the peptides can be incorporatedinto a number of products that can take advantage of the antibacterialproperties that have been discovered for these peptides. Along theselines, the peptides according to the present invention could beincorporated into materials for making surfaces, fibers and films.Examples could include medical devices, such as catheters, endotrachealtubes, scalpel handles, implants, such as hip replacements, kneereplacements, or any other medically related product. Additionalexamples include countertops, cutting boards, sponges, packagingmaterials, wipes, and any number of other articles or mateirals whereantibacterial functionality is desired.

A few examples of polymers that the peptides according to the presentinvention are attached to include polyurethane, polyetherurethane,polyester, silicone, polyamide, polyolefin, polystyrene, polypeptide,polysaccharide, cellulosic, and silk. The peptides of the presentinvention may be linked to the polymer by a non-cleavable linker.Examples of such linkers, as well as polymers and attaching peptides topolymers are provided by U.S. Pat. No. 5,847,047, to Haynie, the entirecontents of the disclosure of which is hereby incorporated by reference.

The β-peptides according to the present invention may also benon-covelently bonded to a carrier. The linking to a carrier may be forthe purpose of facilitating use of a peptide. For example, a peptidecould be combined with a material that could be dried and made into apowder. This powder could then be applied to a countertop material toprovide antibacterial functionality to the countertop. An example of acarrier is sol-gel derived silica glass material.

The peptides according to the present invention could also be used aspart of an antibacterial composition. Along these lines, the peptidescould be incorporated into a solution, foam, or other agent that couldbe used to apply to surfaces for disinfecting purposes. The peptidescould also be utilized with a suitable vehicle or carrier as part of apharmaceutical composition. Such a composition could be appliedtopically or otherwise.

The present invention provides an antibacterial β-peptide having thefollowing formula:

wherein

R₁ is H, an alkyl group including 1-4 carbon atoms, phenyl, heteroaryl,or an alkyl-aryl;

R₂ is an amine-containing alkyl group having the formula —(CH₂)_(m)NH₂,wherein m=1, 2, 3, 4, or 5, (CH₂)_(x)NHC═NHNH₂ wherein x is 1, 2, 3, 4,or 5, a pyridyl, an alkylpryidyl, an amidine-substituted benzyl, aphenyl group, CH₂-imidazole, CH₂-indole or a cyclic amidine;

R₃ is H, an alkyl group including 1-4 carbon atoms, phenyl, heteroaryl,or an alkyl-aryl;

X is —NH₂, —OH, —NHR, or OR where R is alkyl, aryl or acyl groups eitherfree or polymer-supported, a carboxamide, a substituted carboxamide, ora polymer;

Y is H, an alkyl group, an acyl group, an acyl-terminated polymer, asulphonamide, an ether, a urea, a urethane, or a polymer; and

n is 2, 3, 4, 5, 6, or 7.

While the alkyl groups of R₁ and R₃ may be any alkyl group, specificexamples include methyl, ethyl, n-propyl, i-prop, n-butyl, sec-butyl, ortert-butyl.

The present invention also provides an antibacterial P-peptide havingthe following formula:

Y—(T)_(n)—X

wherein

n is 2, 3, 4, 5, 6, or 7;

X is —NH₂, —OH, —NHR, or OR where R is alkyl, aryl or acyl groups eitherfree or polymer-supported, a carboxamide, a substituted carboxamide, ora polymer;

Y is H, an alkyl group, an acyl group, an acyl-terminated polymer, asulphonamide, an ether, a urea, a urethane, or a polymer; and

T is a triplet or triad including T1-T2-T3, wherein T1 comprises ahydrophobic β-amino acid, T2 comprises a polar or basic amino acid, T3comprises either a hydrophobic β-amino acid or a polar or basic β-aminoacid, wherein at least one-half of the triplets include a basic β-aminoacid, in other words, at least one half of the T groups (each includingthree β-amino acids) include a basic amino acid.

Each triplet need not be the same. A peptide could be a homopolymer oftriplets each having the same pattern. Alternatively, a peptide could bea copolymer of repeating patterns of different triplets. For example,two triplets having different amino acid sequence from each other couldbe repeated in the peptide. It can be seen that the peptide couldinclude any repeating pattern of triplets.

According to another formulation, an antibacterial β-peptide has thefollowing formula:

Y-(hydrophobic β-amino acid)-(β-amino acid having a basic sidechain)-(hydrophobic β-amino acid)-X

wherein

X is —NH₂, —OH, —NHR, or OR where R is alkyl aryl or acyl groups eitherfree or polymer-supported, a carboxamide, a substituted carboxamide, ora polymer;

Y is H, an alkyl group, an acyl group, an acyl-terminated polymer, asulphonamide, an ether, a urea, a urethane, or a polymer.

The β-amino acids may be substituted at at least one of the C2 and C3atoms. The substituents may include an aryl, or a C₁₋₁₀ straight orbranched, linear or cyclic alkane, alkene, or alkyne, and wherein thestereochemistry of the P-peptide is in an aldol, or anti configuration.Examples could include —H, —CH₃, —CH(CH₃)₂, —CH₂—CH(CH₃)₂,—CH(CH₃)CH₂CH₃, —CH₂-phenyl, —CH₂-pOH-phenyl, —CH₂-indole, —CH₂—SH,—CH₂—CH₂—S—CH₃, CH₂OH, —CHOH—CH₃, —CH₂—CH₂—CH₂—CH₂—NH₂,—CH₂—CH₂—CH₂—NH—C(NH)NH₂, —CH₂-imidazole, —CH₂—COOH, —CH₂—CH₂—COOH,—CH₂—CONH₂, —CH₂—CH₂—CONH₂, or together with an adjacent —NH group formsa proline amino acid residue. Furthermore, the substiuents on the aminoacids may themselves by substituted. The substituents on thesubstituents may be any of the above-listed substituents on the aminoacids themselves.

Amino acids that are disubstituted have the following configuration:

In other words, both substituents extend toward the same side of themolecule. This configuration typically generates the desired L+2 helicalconfiguration and the desired functionality.

At least one α-amino acid or at least one β-amino acid may be arrangedin the molecule between Y and T and/or X and T. If the amino acid is aβ-amino acid it may be a hydrophobic, polar, or basic β-amino acid.

With either of the above two formulations, the polymers referred to inthe formulas, may be any of the polymers discussed above. Similarly,peptides according to either formulation may be non-covalently bonded toa carrier such as those discussed above.

A particularly stable structure formed by β-peptides is the L₊₂ helixwhich contains three β-amino acid residues per turn of the helix. Thethermodynamic stability of such a helix may also depend upon its chainlength; the longer the chain, the more thermodynamically stable ittypically is. Some β-peptides according to the present invention weredesigned with alternating hydrophobic residues such as homo-valine orhomo-leucine at the i and i+2 positions of the β-peptide with a β-aminoacid at i+1 position having a positively charged amino group on thesidechain such as homo-lysine.

The following represent three examples (I, II, and III) of β-peptidesaccording to a first group:

Fmoc-(β³-HVal-β³-HLys-β³-HLeu)_(n)-OH; n=2-4  (I)

H-(β³-HVal-β³-HLys-β³-HLeu)_(n)-OH; n=2-4  (II)

H-(β³-HLeu-β³-HLys-β³-HLeu)_(n)-OH; n=2-6  (III)

The above peptides were synthesized by solid phase peptide synthesis,using tripeptide blocks prepared on a 2,4-dialkoxy-benzyl ester polymersupport as described below. The Fmoc group in the first series ofpeptides provided a convenient probe for determining the concentrationof the peptides. To determine the effect of this hydrophobic probe onthe properties of the compounds, a parallel series of peptides wassynthesized without this group.

The ability of these peptides to adopt a L+2 helix in aqueous solutionin the presence and absence of micelles and phospholipid bilayers wasassessed using CD spectroscopy, which provides a rapid method to assessthe secondary structure formation of p-peptides. The relationshipbetween secondary structure formation and the CD spectra of peptidescomposed of cyclic β-amino acids is not yet fully developed. However,numerous studies with β-peptides assembled from the acyclic buildingblocks used in this work have demonstrated that the L+2 conformationgives rise to a strong minimum at 215 nm and a maximum at 195 nm in theπ-π* region. The CD spectra of Fmoc-(β³-HVal-β³-HLys-β³-HLeu)_(n)-OH(n=2-4) in aqueous solution failed to exhibit these features associatedwith the L+2 helical conformation. This finding is consistent withprevious studies showing that complete formation of the L+2 conformationby β-peptides of this length requires the addition of organic solventsand/or conformationally constrained amino acids. However, the additionof dodecyl phosphocholine (DPC) micelles resulted in a length-dependentincrease in the magnitude of [θ]_(215 nm), reaching an intensityconsistent with essentially complete helix formation at n=4. Similardata were observed with small, unilamellar vesicles composed of POPC(not shown). Hydrophobic/water interfaces are similarly able to induceα-helix formation in a variety of amphiphilic α-peptides.

The presence of the hydrophobic Fmoc group appears to favor binding toDPC micelles (and concomitant formation of an L+2 helical conformation),based on the slightly greater negative ellipticity at 215 nm observedfor Fmoc-(β3-HVal-β³-HLys-β³-HLeu)₃-OH versusH-(β³-HVal-β³-HLys-β³-HLeu)₃-OH. The dependence of amino acidcomposition on L+2 helix formation was probed by comparing the CDspectra of H-(β³-HVal-β³-HLys-β³-HLeu)₃-OH versusH-(β³-HLeu-β³-HLys-β³-HLeu)₃-OH. These spectra indicate that theβ³-HVal-containing β-peptide has a slightly greater propensity to formthe L+2 helix than the β³-HLeu-containing peptide. However, at chainlengths of 12 residues (n=4) or longer, helix formation appeared to becomplete for all three series of peptides, because further chainelongation failed to increase the intensity of [θ]_(215 nm).

The biological activities of these peptides were measured using humanerythrocytes and E. coli as models for mammalian and bacterial cells,respectively. Hemolysis was monitored in 10 mM Tris, 150 mM NaCl, pH7.0, while the bacterial assay was conducted in minimal media M9 (Table1); both of these solutions are sufficiently transparent to allow CD andultracentrifugation measurements (supplementary material). Under theassay conditions, the peptides gave CD spectra similar to those in Trisbuffer, with the exception of H-(β³-HLeu-β³-HLys-β³-HLeu)_(n)-OH, n=5and 6. Analytical ultracentrifugation indicated that these two peptidesformed large aggregates in the presence of phosphate (an essentialcomponent of minimal media).

All three series of peptides show length-dependent antibacterialactivities, which correlates with their helical contents in DPCmicelles. In DPC micelles the helical content of the three 9-residueβ-peptides follow the progressionFmoc-(β³-HVal-β³-HLys-β³-HLeu)₃-OH>H-(β³-HVal-β³-HLys-β³-HLeu)₃-OH>H-(β³-HLeu-β³-HLys-β³-HLeu)₃-OH.Approximately the same trend is observed in the biological data.However, the biological activities of the peptides continue to increaseas their chain lengths are increased beyond the threshold required forcomplete helix formation in DPC micelles. This result may reflect thefact that longer helices have a higher surface area available forbinding to membrane surfaces, providing enhanced affinity and greaterefficacy.

Below are two more examples (IV and V) of the first group of peptides.These are particular examples of the molecules shown above, withparticular ranges for n.

β-amino acids IV and V were prepared using the Arndt-Eisterthomologation of N-Fmoc α-amino acids as described by Seebach asdescribed in International Patent Document WO 97/47593 to Seebach, theentire contents of the disclosure of which are hereby incorporated byreference, and peptides were prepared via solid-phase peptide synthesis(Fmoc chemistry) in tripeptide blocks. The peptides greater than 6residues (2 turns of the L₊₂ helix) were found to have length-dependentantibacterial activities TμM levels) against E. coli. These moleculesalso exhibit hemolytic activity against human red blood cells.

Stress-induced response and growth inhibition assays were carried out onsamples of four of the β-peptide and compared to known polycationicpeptides. The results are listed in Table 1.

TABLE 1 Stress Response and Growth Inhibition Results of β-peptidesosmY- micF- MIC* natural peptides lux lux (μM) Polymyxin B + + 0.2Polymyxin E + + 0.15 Cecropin A + + 0.1 Cecropin B + + 0.1 Magainin 1 +− 12 Magainin 2 + − 5 ≈IC₅₀ of recA-lux inhibi- tion # IC₅₀− HD50/β-peptides (μM) (μM) HD50+ IC50 Fmoc-(hVal- − − 16 15 6.3 0.42hLys-hLeu)₃-OH H-(hLeu-hLys- − − 64 1.7 2.6 1.5 hLeu)₄-OHH-(hLeu-hLys- + − 24 ND 0.23 hLeu)₅-OH H-(hLeu-hLys- − − 2 ND 0.081hLeu)₆-OH

Polymyxin B was tested both rich, LB, medium and defined, M9, medium.The other natural peptides were tested in LB medium.

The β-peptides were tested in M9 medium. * In LB medium (data from Oh etal. Biochem. Biophys. Acta. 2000,1463: 43-54)

∇in M9 medium (data from Hamuro et al., ref. 5) # In M9medium.+Hemolysis experiments were performed by incubating a 0.25%suspension of human RBc's in 10 mM Tris buffer containing 150 nM NaCl atpH 7.0 with varying amounts of peptide. The hemolytic dose required tolyse 50% of the RBC's is reported as the HD₅₀.

With the exception of H-(hLeu-hLys-hLeu)₅-OH, the β-peptides did notinduce the osmY-lux fusion, an observed induction for polycationicamphiphilic antibacterial α-peptides such as the magainins. However,similar to the magainins, no induction of the micF-lux fusion wasobserved. Also, the minimum inhibitory concentrations (MIC's) weredetermined to be higher than those reported in the published manuscript.Results from these assays can aid in understanding of the geneticprofile of antibacterial activities displayed by these and otherβ-peptides.

Table 2 below provides selected data regarding the β-peptides of group1.

TABLE 2 Antibac- Selec- terial tivity Hemolysis^(a) assay^(b) HD50/Peptide HD50 (μM) IC50 (μM) IC50Fmoc-(β³-HVal-β³-HLys-β³-HLeu)₂- >100 >100 — OHFmoc-(β³-HVal-β³-HLys-β³-HLeu)₃- 6.3 15 0.42 OHFmoc-(β³-HVal-β³-HLys-β³-HLeu)₄- 0.31 1.5 0.21 OHH-(β³-HVal-β³-HLys-β³-HLeu)₂-OH >100 >100 —H-(β³-HVal-β³-HLys-β³-HLeu)₃-OH 86 41 2.1H-(β³-HVal-β³-HLys-β³-HLeu)₄-OH 4.2 2.1 2.0H-(β³-HLeu-β³-HLys-β³-HLeu)₂-OH >100 >100 —H-(β³-HLeu-β³-HLys-β³-HLeu)₃-OH >100 35 >2.9H-(β³-HLeu-β³-HLys-β³-HLeu)₄-OH 2.6 1.7 1.5H-(β³-HLeu-β³-HLys-β³-HLeu)₅-OH 0.23 — — H-(β³-HLeu-β³-HLys-β³-HLeu)₆-OH0.081 — — ^(a)Hemolysis experiments were performed by incubating a 0.25%suspension of human erythrocytes (RBC's) in 10 mM Tris buffer containing150 mM NaCl at pH 7.0 with varying amounts of peptide. After 1 h at 37°C., the suspensions were centrifuged and the OD_(414 nm) of thesupernatant (due to released hemoglobin) was measured. The hemolyticdose required to lyse 50% of the RBC's was obtained as described in theSupplementary Material. ^(b)Antibacterial assays were performed byincubating varying amounts of peptide with cultures of K91 E. Coli inminimal media at pH 7.4. After 9 h at 37° C., the OD_(600 nm) of theculture was measured (light scattering due to bacteria). The peptidedose required to supress 50% bacterial growth was obtained as describedherein.

(a) Synthesis of Fmoc-β³-HXxx-β³-HLys(Boc)-β³-HLeu-OH. The protectedpeptide fragments were assembled by using super acid labile HMPB-MBHAresin (Scheme 1). First, Fmoc-β³-HLeu-OH was attached onto the resin byDIC-DMAP protocol followed by pivaloyl anhydride capping. A standardFmoc solid phase peptide synthesis, deprotection of Fmoc group bypiperidine and coupling with Fmoc-β-amino acids with HBTU-HOBt, gave thetripeptides on the resin, which were then treated with 1% TFA indichloromethane to give the desired protected β-peptide fragments in(HXxx=HVal, 56%; HXxx=HLeu, 86%). The crude peptide fragments were usedfor the segment condensation without further purification.

(b) Synthesis of Fmoc-(β³-HValβ³-HLys-β³-HLeu)_(n)-OH (n=2-4) andH-(β³-HLeu-β³-HLys-β³-HLeu)_(n)-OH (n=2-6). The titled β-peptides weresynthesized from protected tripeptide fragment,Fmoc-β³-HXxx-β³-HLys(Boc)-β³-HLeu-OH (HXxx=HVal or HLeu), using Wangresin and standard Fmoc protocol (Scheme 2). The first fragment wasattached onto Wang resin by DIC-DMAP protocol and the unreacted hydroxylgroup on the resin was capped by the treatment with a solution ofbenzoyl chloride and pyridine. The reiterative piperidine deprotectionsand HBUT-HOBt couplings gave the β-peptides on Wang resin. TFA cleavageand HPLC purification yielded the titled β-peptides.

The following describes the synthesis of β-peptides of the Group I.Fmoc-(b³-HVal-b³-HLys-b³-HLeu)_(n)-OH (n=2-4) andFmoc-(b³-HLeu-b³-HLys-b³-HLeu)_(n)-OH (n=2-6) were prepared by segmentcondensation from the fully protected tripeptide fragmentsFmoc-(b³-HVal-b³-HLys(Boc)-b³-HLeu)-OH andFmoc-(b³-HLeu-b³-HLys(Boc)-b³-HLeu)-OH, respectively. The initialtripeptide fragment was coupled to Wang resin employingdiisopropylcarbodiimide (DIC)/4-dimethylaminopyridine (DMAP) activation.Subsequent tripeptide fragments were added sequentially after terminalFmoc-deprotection (20% piperidine in N,N-dimethylformamide (DMF))employing 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU)/1-Hydroxybenzotriazole (HOBt) activation. Theresin bound peptides were cleaved and side-chain deprotected (50%trifluoroacetic acid (TFA) in CH₂Cl₂).

The peptides H-(b³-HVal-b³-HLys-b³-HLeu)_(n)-OH (n=2-4) andH-(b³-HLeu-b³-HLys-b³-HLeu)_(n)-OH (n=2-6) were prepared by N-terminalFmoc-deprotection (20% piperidine in DMF) ofFmoc-(b³-HVal-b³-HLys-b³-HLeu)_(n)-OH (n=2-4) andFmoc-(b³-HLeu-b³-HLys-b³-HLeu)_(n)-OH (n 2-6).

Tripeptide fragments Fmoc-(b³-HVal-b³-HLys(Boc)-b³-HLeu)-OH andFmoc-(b³-HLeu-b³-HLys(Boc)-b³-HLeu)-OH were prepared on super acidlabile HMPB-MBHA resin by standard Fmoc solid phase peptide synthesisprotocol employing HBTU/HOBt activation. Treatment of the resin-boundpeptide with 1% TFA in CH₂Cl₂ afforded fully protected crude tripeptidewhich was used for segment condensations without further purification.

The appropriate Fmoc-protected b-amino acid residues used to synthesizethe tripeptide fragments were prepared from the correspondingFmoc-a-amino acids by Arndt-Eistert homologation following the proceduredisclosed by Atherton, E.; Sheppard, R. C. In The Peptides; Udenfriend,S., Meienhofer, J., Ed.; Academic Press: Orland, Fla., 1987; Vol. 9; pp1-38, the entire contents of the disclosure of which is herebyincorporated by reference. The synthesis of all tripeptide fragments andfull length peptides are outlined below.

Fmoc-a-amino acids were purchased from Bachem Bioscience Inc., HBTU fromAdvanced ChemTech., and all other reagents from Aldrich and were usedwithout further purification. HMPB-MBHA (loading=0.51 mmol/g) and Wang(loading=0.85 mmol/g) resins were purchased from Novabiochem. Peptideswere synthesized in standard glass peptide synthesis vessels. Nominalmass determinations were carried out by electrospray ionization (ESI)employing a Hewlett-Packard 1100 coupled to a Micromass Platform LC.High resolution mass spectra were obtained with a Micromass AutoSpec ESImass spectrometer. UV measurements were performed with a Hewlett-Packard8453 spectrometer. Peptides were purified using a preparative Vydac C4peptide/protein column. Solvent A was composed of water and 0.1% TFA andsolvent B was composed of 90% acetonitrile, 10% water and 0.1% TFA,unless noted otherwise. Peptide purity was accessed by analytical HPLCanalyses employing a HP-1100 liquid chromatography system equipped witha photodiode array detector and a Vydac C4 column (2.1×150 mm).

General Procedure for the synthesis of tripeptide fragmentsFmoc-(b³-HVal-b³-HLys(Boc)-b³-HLeu)-OH andFmoc-(b³-HLeu-b³-HLys(Boc)-b³-HLeu)-OH. HMPB-MBHA resin (5.0 g, 2.55mmol) was added to a solution of Fmoc-b³-HLeu-OH (1.10 g, 3.0 mmol), DIC(470 mL, 3.0 mmol) and DMAP (0.37 g, 3.0 mmol) in CH₂Cl₂ (50 mL) and thereaction mixture was left at room temperature for 40 h. The resin wasthen washed with DMF (4×1 min). Unreacted resin was capped with asolution of trimethylacetic anhydride (5.2 mL, 25.5 mmol) and pyridine(2.06 mL, 25.5 mmol) in DMF (40 ML) for 1 h and the resin subsequentlywashed with DMF (5×1 min). The following cycle was used to couplesubsequent Fmoc-protected amino acids: Fmoc-deprotection (20% piperidinein DMF for 1 h), DMF wash (5×1 min), coupling (2.8 mmol of Fmoc-b-aminoacid, 1.06 g (2.8 mmol) of HBTU, 0.43 g (2.8 mmol) of HOBt, 0.98 mL (5.6mmol) of diisopropylethylamine (DIEA) in DMF (40 mL) for 40 h), DMF wash(5×1 min).

After the final coupling, the resin was washed with DMF (5×1 min), MeOH(4×1 min), CH₂Cl₂ (4×1 min) and dried. The resin-bound tripeptide wascleaved with 1% TFA in CH₂Cl₂ (50 mL×8 min, 50 mL×2 min×4). Eachfiltrate was directly introduced into 2% pyridine in MeOH (50 mL). Theresin was washed with CH₂Cl₂ (3×50 mL×5 min) and MeOH (3×50 mL×5 min).All filtrates were combined and the volume reduced by evaporation invacuo to a final weight of 60 g. Water (150 mL) was then added toprecipitate the desired fully protected tripeptide which was collectedby filtration and dried under high vacuum affording product as a whitepowder. Crude peptide fragments were used for segment condensationswithout further purification.

Fmoc-b³-HVal-b³-HLys(Boc)-b³-HLeu-OH. White solid (593 mg, 56%): HRMS(ESI) m/e calcd for C₄₀H58N₄O₈Na(M+Na⁺) 745.4152, found 745.4136.

Fmoc-b³-HLeu-b³-HLys(Boc)-b³-HLeu-OH. White solid (1.61 g, 86%): HRMS(ESI) m/e calcd for C₄₁H₆₀N₄O₈Na (M+Na⁺) 759.4309, found 759.4335.

General Procedure for the synthesisFmoc-(b³-HVal-b³-HLys-b³-HLeu)_(n)-OH (n=2-4). Wang resin (0.88 g, 0.75mmol) was added to a suspension ofFmoc-(b³-HVal-b³-HLys(Boc)-b³-HLeu)-OH (0.5 mmol), DIC (78 mL, 0.5 mmol)and DMAP (0.06 g, 0.5 mmol) in CH₂Cl₂ (15 mL) and the reaction mixturewas left at room temperature for 48 h. The resin was then washed withCH₂Cl₂ (4×1 min), DMF (4×1 min), MeOH (4×1 min), CH₂Cl₂ (4×1 min).Unreacted resin was capped with a solution of trimethylacetic anhydride(0.5 mL, 2.5 mmol) and pyridine (0.2 mL, 2.5 mmol) in DMF (10 mL) for 2h and the resin subsequently washed with DMF (5×1 min). The followingcycle was used to couple subsequent tripeptide fragments (aliquots ofresin-bound peptide were removed after each coupling step to obtainresin-bound peptides of appropriate length): Fmoc-deprotection (20%piperidine in DMF for 1 h), DMF wash (4×1 min), MeOH wash (4×1 min),CH₂Cl₂ wash (4×1 min), coupling (1.2-fold excess of tripeptide, HBTU,HOBt, and two-fold excess of diisopropylethylamine (DIEA) in DMF for 10h), DMF wash (4×1 min), MeOH wash (4×1 min), CH₂Cl₂ wash (4×1 min).After the final coupling, the resin was washed with DMF (5×1 min), MeOH(4×1 min), CH₂Cl₂ (4×1 min) and dried. The resin-bound peptide wascleaved and side-chain deprotected with 50% TFA in CH₂Cl₂ for 1 h andthe resin washed with CH₂Cl₂ (3×25 mL). The combined filtrates wereevaporated affording an oil which was triturated with cold diethyl etheryielding crude Fmoc-(b³-HVal-b³-HLys-b³-HLeu)_(n)-OH (n=2-4) which wassubsequently purified by HPLC.

Fmoc-(b³-HVal-b³-HLys-b³-HLeu)2-OH. Purification by preparative HPLCemploying a linear gradient from 30% to 60% solvent B over 60 min. HRMS(ESI) m/e calcd for C₅₅H₈₉N₈O₉ (M+H⁺) 1005.6753, found 1005.6748.

Fmoc-(b³-HVal-b³-HLys-b³-HLeu)₃-OH. Purification by preparative HPLCemploying a linear gradient from 50% to 80% solvent B over 60 min. HRMS(ESI) m/e calcd for C₇₅H₁₂₇N₁₂O₁₂ (M+H⁺) 1387.9696, found 1387.9853.

Fmoc-(b³-HVal-b³-HLys-b³-HLeu)4-OH. Purification by preparative HPLCemploying a linear gradient from 60% to 75% solvent B over 60min.(solvent B is composed of 60% acetonitrile, 30% isopropyl alcohol,10% water and 0.1% TFA). FIRMS (ESI) m/e calcd for C₉₅H₁₆₅N₁₆O₁₅ (M+H⁺)1770.2640, found 1770.2777.

General Procedure for the synthesis H-(b³-HVal-b³-HLys-b³-HLeu)_(n)-OH(n=2-4). An eppendorf tube was charged with 2 mgFmoc-(b³-HVal-b³-HLys-b³-HLeu)_(n)-OH and 0.25 mL of 20% piperidine inDMF. After 30 min., the solution was evaporated in vacuo yielding anoil. The oil was then triturated with cold diethyl ether affording awhite solid which was collected by filtration, washed with cold diethylether (3×1 mL) and dried.

H-(b³-HVal-b³-HLys-b³-HLeu)2-OH. LRMS (MALDI-TOF) m/e calcd forC₄₀H₈₀N₈O₇ (M+H⁺) 784.61, found 784.28.

H-(b³-HVal-b³-HLys-b³-HLeu)3-OH. LRMS (MALDI-TOF) m/e calcd forC₆₀H₁₁₈N₁₂O₁₀(M+H⁺) 1166.9, found 1167.2.

H-(b³-HVal-b³-HLys-b³-HLeu)4-OH. LRMS (MALDI-TOF) m/e calcd forC₈₀H₁₅₆N₁₆O₁₃ (M+H⁺) 1549.2, found 1549.6.

General Procedure for the synthesis H-(b³-HLeu-b³-HLys-b³-HLeu)_(n)-OH(n=2-6). These peptides were synthesized in a similar manner asdescribed above with the exception that the N-terminally Fmoc-protectedpeptides (Fmoc-(b³-HLeu-b³-HLys-b³-HLeu)_(n)-OH) were not purified priorto the final Fmoc-deprotection step. Purification and mass spectral dataof the titled peptides are as follows:

H-(b³-HLeu-b³-HLys-b³-HLeu)₂-OH. Purification by preparative HPLCemploying a linear gradient from 20% to 50% solvent B over 60 min. HRMS(ESI) m/e calcd for C₄₂H₈₂N₈O₇ (M+H⁺) 811.6385, found 811.6345.

H-(b³-HLeu-b³-HLys-b³-HLeu)₃-OH. Purification by preparative HPLCemploying a linear gradient from 30% to 60% solvent B over 60 min. LRMS(ESI) m/e calcd for C₆₃H₁₂₃N₁₂O₁₀ (M+H⁺) 1208.0010, found 1208.0.

H-(b³-HLeu-b³-HLys-b³-HLeu)₄-OH. Purification by preparative HPLCemploying a linear gradient from 40% to 70% solvent B over 60 min. LRMS(ESI) m/e calcd for C₈₄H₁₆₃N₁₆O₁₃ (M+H⁺) 1604.2586, found 1604.2666.

H-(b³-HLeu-b³-HLys-b³-HLeu)5-OH. Purification by preparative HPLCemploying a linear gradient from 45% to 75% solvent B over 60 min. LRMS(ESI) m/e calcd for C₁₀₅H₂₀₂N₂₀O₁₆ (M) 1999.6, found 1001.3 (M+2^(H+)),668.2 (M+3H⁺), 501.5 (M+4H⁺).

H-(b³-HLeu-b³-HLys-b³-HLeu)6-OH. Purification by preparative HPLCemploying a linear gradient from 50% to 80% solvent B over 60 min. LRMS(ESI) m/e calcd for C₁₂₆H₂₄₂N₂₄O₁₉ (M) 2396.0, found 1199.6 (M+2H⁺),800.3 (M+3H⁺), 600.6 (M+4H⁺).

Circular Dichroism spectra were obtained on an AVIV 62DSspectropolarimeter using 1 mm quartz cells. Samples were prepared fromstock solutions in either water or 50 mM Tris buffer (pH 7) and dilutedto the desired concentration with the appropriate buffer or buffercontaining lipid. Peptide concentrations were determined from dry weightusing freshly lyophilized samples. Molecular weights were calculated,assuming that the peptides bound one TFA counterion per amine. Theconcentration was also determined for Fmoc-containing peptides from theabsorbance of the fluorenyl group. Good (within ±10%) agreement wasobserved between the two methods. All spectra are corrected for buffercontributions and are reported in units of mean residue ellipticity. TheCD spectra revealed that the peptides form L+2 helices when they bind tomicelle and bilayer surfaces. The formation of this structure correlateswith their hemolytic and antibacterial activities.

The amphiphilic β-peptides of Group I are highly active against E. colias well as red cells. The potency of the longest peptide (80 nM) isconsiderably greater than that of melittin, whose LD₅₀ is approximately0.5 μM under these conditions. However, the selectivity of the currentβ-peptides for bacterial versus mammalian cells is low.

With the above specific antibacterial β-peptides, the large hydrophobicgroups from the homo-leucine i position of the β-peptide may beresponsible for the observed hemolytic activity. By comparison, with thespecific antibacterial β-peptides illustrated below, the hydrophobicityat the i position of the peptide was reduced by using homo-alanineinstead of homo-leucine. The formula below represents the structure ofmembers of a second group of peptides according to the presentinvention, which will referred be to herein as peptides 1 and 2 of thesecond group. (group II).

As described below in greater detail, peptides 1 and 2 both adopt L₊₂helix conformation upon binding to lipid micelle or vesicle. They havehigh antibacterial activity against bacteria with IC50 of several μMs,and low toxicity towards mammalian cells.

As mentioned above, for this and other proteins and peptides, circulardichoism (CD) spectroscopy is a fast and easy way to determine secondarystructure. For peptides of β-amino acids, the standard of structuredetermination is not as well developed as it is for peptides derivedfrom a-amino acids. Nevertheless, typical CD spectra of several types ofhelices adopted by β-peptides have been reported. The CD spectra of L₊₂helix have been demonstrated experimentally to have a positive cottoneffect centered around 195 nm and a negative one centered around 215 nmarising from exton π-π* transition. The CD spectra of peptides 1 and 2of Group II were measured with and without the presence of dodecylphosphocholine (DPC) micelle. FIG. 1 illustrates the results of thesetests.

In aqueous solution without lipid micelle, the CD spectra of bothpeptides are quite flat, suggesting random conformations. With 5 mM DPC,strong positive peak around 195 nm and negative peak around 215 nm areobserved, indicating formation of L₊₂ helix. The magnitudes of meanresidue ellipticities of 1 and 2 are very close, suggesting the helixformation is complete at n=4. The same results are obtained with thestructures above that displayed the hemolytic properties. The positionof the minimum shifts slightly with the peptide length grows. Theminimum of 2 is centered at 213 nm, while it is centered at 214 nmfor 1. The CD data agree with the results. Without the lipid micelles,the peptides tend to be random coil because of the repulsion amongpositive charged homo-Lys side chains. With the presence of micelle, thehydrophobic side chains assembles to the micelle surface and theamphiphilic L₊₂ helix forms.

These two β-peptides showed greater selectivity towards antibacterialactivity against E coli with IC_(50's) in the low micromolar range butmuch less hemolytic than the antibacterial β-peptides first describedabove. Table 3 demonstrates the properties for peptides 1 and 2 above.

TABLE 3 n = 4 n = 5 IC₅₀ (μmol)*  6  4 IC₅₀ (μmol)#  10  3 HD₅₀ (μmol)670 240 HD50/IC50 111  60 induction of osmY-lux + + induction ofmicF-lux + + *K91 E. coli and peptide of different concentrations inminimal media, at pH 7.4 (DeGrado lab) #inhibition of recA-luxbioluminescence in M9 media (Van Dyk lab).

Samples of these β-peptides 1 and 2 were evaluated in the stress-inducedresponse assays. Unlike the previous antibacterial β-peptides, bothshowed an induction of the osm-Y and mic-F fusions similar to thececropins and polymyxins, as shown in Table 3.

The biolumenescence data result from test carried out according to U.S.Pat. No. 5,683,868, Highly Sensitive Method For Detecting EnvironmentalInsults, issued Nov. 4, 1997 to LaRossa et al., the entire contents ofthe disclosure of are hereby incorporated by reference, The testsdescribed in this patent permit antibacterial properties of peptides orany other materials to be assessed. FIGS. 5 and 6 illustrate testresults demonstrating the antibacterial properties of the peptides.Along these lines, FIG. 5 illustrates a relationship between relativelight units and minutes of exposure of bacteria to the various materialsin the legend. P×B represents polymyxin, a known antibacterial material.In the graph, a decrease in RULs corresponds to a decrease in bacteria.As can be seen in FIG. 5, a peptide according to the present invention,where n is 5 produces similar results to the P×B. FIG. 6 illustrates theresults of another study, These graphs demonstrate the antibacterialproperties of the peptides. Table 4 below summarizes the results of someof these tests.

TABLE 4 Gene Stress Regulatory Osmotic (hAla-hLys- (hAla-hLys- fusionsensed gene shock (NaCl) Polymyxin hVal)₄ hVal)₅ micF N/A Rob yes yesyes yes osmY osmotic (not ROB) yes yes yes yes shock

Experimental Methods And Materials For Group II Peptides 1 and 2

Fmoc α-amino acid pentafluorophenyl esters were purchased from NovaBiochem, HBTU and HOBt from Advanced ChemTech., Fmoc PAL-PEG-PS resin(loading 0.17 mmol/g) from PerSeptive Biosystems, SOPC and SOPS fromAvanti Polar-Lipids, Inc., Calcein from Lancaster Synthesis, TritonX-100 from, and all other reagents from Aldrich and were used withoutfurther purification. All Fmoc,-amino acids were synthesized from thecorresponding Fmoc α-amino acid pentafluorophenyl esters viaArnst-Eistert homologation following Seebach's published procedure (seeInternational Patent Document WO 97/47593 to Seebach). Peptides weresynthesized in a standard glass peptide synthesis vessel. Thepurification was carried out on a Waters HPLC using a Vydac C4 column.Solvent A was composed of 1% TFA in water a n d solvent B was composedof 90% acetonitrile, 10% water and 0.1% TFA. Mass spectra were measuredon a Hewlett-Packard 1100 ESI spectrometer and a PerSpective BiosystemsVoyager-DERP MALDI-TOF mass spectrometer. NMR spectra were obtained on aBruker AC-250 spectrometer. UV-Vis spectra were measured on aHewlett-Packard 8453 spectrometer. Fluorescence measurement was carriedout on a a Hitachi F-2500 Fluoresence spectrophotometer. CD spectra wereobtained on an AVIV 62DS spectropolarimeter.

Synthesis Of H-(HAla-HLys-HVal)_(n)—NH₂ (n=4, 5)

Fmoc PAL-PEG-PS resin (588 mg, 0.1 mmol) was swell in DMF (5 mL) for 30min before the synthesis. The Fmoc was deprotected with 20%piperidine/DMF (3×5 mL x 5 min), and washed with DMF (5×5 mL x 2 min).Amino acid coupling was carried out by adding 2 mL solution of aminoacid (0.25 mmol), HBTU (95 mg, 0.25 mmol), HOBt (34 mg, 0.25 mmol), DIEA(139 μL, 0.8 mmol) in DMF to the resin, shaking for 4 h, and washingwith DMF (5×5 mL×2 min). The peptides were cleaved from the resin bytreatment of TFA/TIS (95:5) for 2 h. The solution was concentrated andthe peptide was precipitated out with addition of cold ether. Peptideswere purified by HPLC on a reverse phase C4 column, with a lineargradient from 20% to 50% solvent B in 50 min for n=4, and from 30% to60% solvent B in 60 min for n=5. LRMS (MALDI-TOF) m/e calculated for n=4C₆₈H₁₃₂N₁₇O₁₂ (MH+) 1379.1, found 1379.6. LRMS (MALDI) m/e calculatedfor n=5 C₈₅H₁₆₄N₂O₁₅ (MH⁺) 1719.4, found 1720.1.

Circular Dichroism Studies

CD spectra were measured on an AVIV 62DS spectropolarimeter using both 1mm and 10 mm quartz cuvettes. Sample stock solutions were prepared inwater and diluted into appropriate buffers. Peptide concentrations weredetermined from the dry weight of lyophilized peptide, and werecalibrated by the UV absorbance of Fmoc before the deprotection.

Hemolysis Assay

Hemolysis experiments were carried out by incubating 0.25% suspension ofhuman erythrocytes (RBC's) with peptides of different concentrations in150 mM sodium choloride, 10 mM Tris buffer, pH 7.0. The sample wasprepared by combining 400 μL of the RBC suspension and 100 μM of thepeptide solution. After incubated the sample at 37_(—) C. for 1 hour,the sample was centrifuged at 14,000 rpm for 5 min, and the OD₄₁₄ of thesupernatant was measured. Mellitin (50 μM) was used to get 100%hemolysis. The hemolytic IC50 was obtained by plotting hemolysispercentage vis. peptide concentration, and the concentration requiredfor 50% hemolysis was determined from the smooth curve-fitted graph.

Antibacterial Assay

Antibacterial assay was performed by incubation of K 91 E. coli andpeptide of different concentrations in minimal media, at pH 7.4. Thepeptide solution (50 μL) and K 91 E. coli culture (20 μL, grown inminimal media for 24 hr-36 hr) were mixed with 1 mL minimal media. Afterincubation at 37° C. for 8 hr, the OD₆₀₀ was measured. The peptide doserequired for 50% suppress of bacterial growth was obtained from thesmooth fitted OD₆₀₀ vs. log[peptide] curve.

Peptide Binding to Phospholipid Bilayers

The binding affinity of peptides to phospholipid bilayers was measuredusing CD spectra of the peptide in the presence of varying lipid vesicleconcentrations. Peptides show little structure from CD spectra in water.Upon addition of lipid vesicles, the ellipiticity at 214 nm increased asa consequence of helix formation due to interactions between the peptideand lipid surface. Small unilamellar vesicles (SUV) of SOPC/SOPS wereprepared by sonicating lipid large vesicles in 10 mM phosphate buffer,pH 7. CD spectra were taken before and after the addition of aliquots ofvesicle to 2 mL peptide solution in 10 mM phosphate buffer, pH 7.

The dissociation constant Kd was determined from equation (1) for singlesite binding:

[P](R−1)−c/n−Kd+c/(nR)=0  (1)

where [P] and c are peptide and lipid molar concentrations,respectively, n is the number of lipids per peptide binding site. R isthe fraction of peptide bound, which can be calculated from equation(2):

R=(θ_(c)−θ₀)/(θ₈−θ₀)  (2)

where θ_(c) and θ₀ are the ellipticities at 214 nm at lipidconcentration of c and o, respectively, θ_(max) represents theellipticity at 214 nm of saturated binding. Since lipid vesicles causelight scattering, it was difficult to obtain accurate values for θ₈.Therefore θ₈ was considered as a variable during the curve fitting.Curve fitting was done by using KaleidaGraph program.

Peptide-Induced Leakage of Liposomal Contents

The leakage of liposome contents to the external media was monitored bythe release of calcein encapsulated in vesicles. The vesicle wasprepared by reverse-phase evaporation in 10 mM sodium phosphate buffer,pH 7, followed by single extrusion through one 0.2 μm pore sizepolycarbonate filter. The non trapped calcein was removed by elutingthrough a size exclusion Sephadex G-25 column, with 90 mM sodiumchloride, 10 mM sodium phosphate, pH 7. The leaking kinetics wasmonitored by follow the increase of calcein fluorescence intensity at520 nm (excitation at 490 nm) due to release of self-quenching. Theexperiment was carried out on a Hitachi F-2500 Fluoresencespectrophotometer, with a slit width of 5 μm. The initial rate wascalculated from the linear part of the %leakage vs. time curve.

Antibacterial Assay and Hemolysis Experiment

The activity and selectivity of peptide 1 and peptide 2 were examined byantibacterial assay and hemolysis experiments. E. Coli and humanerythrotytes (RBC) were used as models for bacterial cell and mammaliancell, respectively. The peptide concentration required for 50% bacterialgrowth suppression (IC50), and peptide concentration required for 50%RBC lysis (HD50) are listed in Table 3. Both peptides show highantibacterial activity with IC50 of several μMs, which is comparable tothe natural antibacterial peptide magainin (MIC3.2 μg/ml), and the“β-17” beta peptide (MIC6.3 μg/ml) recently reported from Gellman andcoworkers (see Porter et al., NATURE, 2000, 404, 565). The antibacterialactivities of peptide 1 and peptide 2 are about the same except peptide2 has slightly lower IC50. Similar trend has been observed in theprevious study, that longer peptide tends to have higher activity.However, the difference in IC50 is much smaller for this series ofpeptide than the previously studied ones. Antibacterial peptide withgood selectivity is usually represented by its low hemolysis ability.Our results from hemolysis experiment indicate that selectivity of thisseries of peptide is greatly improved compared to the previous studiedones. The HD50 is 670 μM for peptide 1 and 240 μM for peptide 2, whichgives a selectivity of 111 for peptide 1 and 60 for peptide 2.

TABLE 5 Peptide 1 (n = 4) Peptide 2 (n = 5) IC50 6 4 HC50 670 240selectivity 111 60 Kd (SOPS/SOPC (1:9)) 1.4 0.3 Kd (SOPC) — 15

Membrane Binding Studies

To get better understanding of the binding mechanism of the peptides tolipid membranes, the dissociation constants (Kd) of peptide 1 andpeptide 2 to small unilamellar lipid vesicles (SUV) were measured. Sincebinding to DPC micelle drives peptide 1 and peptide 2 from random coilconformation to helical conformation, the binding equilibrium can bemonitored by CD spectroscopy. The binding curve obtained from CDmeasurement is shown in FIG. 2, and the dissociation constant Kd derivedfrom curve fitting with single site binding equation is listed inTable 1. Small unilamellar vesicles (SUV) were used as lipid bilayermodel. Mammalian cell membranes mainly consist of charge neutralphosphocholin, while bacterial outer membranes have more negativecharges coming form lipopolysaccharide or presence of teichoic andteichuronic acids, and of amino acid carboxyl groups in the multilayeredpeptidoglycan. In order to mimic bacterial, 10% negative chargedphosphoserine (SOPS) was mix with SOPC to generate SUVs. Both peptidesexhibit tight binding to SOPS/SOPC (1:9) vesicles. With an equivalenceof 42 lipid molecules per peptide molecule, a Kd of 1.4 μM was obtainedfor peptide 1. For each peptide 1, the surface area for binding is 188Å², the corresponding area access for binding from the outer leaflet ofthe lipid bilayer is 65 Å²×21=1365 Å². Thus the ratio of areas is 7.3:1(lipid:peptide). Presumably, the ratio is the same for both peptide 1and peptide 2. Using an equivalence number of 52 for peptide 2, a Kd of0.3 μM was obtained for SOPS/SOPC (1:9). The longer peptide bindstighter than the shorter one. But the difference is small, just like theIC50 values of peptide 1 and peptide 2. Vesicles of phosphocholin (POPCand SOPC) were used as models of mammalian cell membrane, peptide 1 doesnot show any formation of helix while peptide 2 shows increase of helixcontent. The Kd of peptide 2 to SOPC LUV is 15 μM. The much loweraffinity of peptide 2 to SOPC vesicles than to SOPS/SOPC (1:9) vesiclesexplains its selective activity against bacterial cells. The failure tobind SOPC vesicles of peptide 1 elucidates its better selectivity thanpeptide 2.

Peptide-Induced Leakage of Liposomal Contents

The mechanism of how the antibacterial peptides work is still on debate.However, it is generally believed they kill the cell by disturbing cellmembranes. The experiment of peptide-induced leakage of liposomalcontents is a direct way to monitor this process. Large unilammilarvesicles (LUV) of SOPS/SOPC (1:9) were used in the binding study. Theleakage of encapsulated calcein was detected by its fluorescence at 515nm. The leakage percentage vs. time is shown in FIG. 3. The percentageof leakage at 285 seconds was plotted vs. peptide concentration and wasshown in FIG. 4. The fraction of bound lipid at different peptideconcentration was calculated and overlaid in FIG. 4 as well. The lysisabilities of the two peptides are similar at low peptide concentration,and become quite different with increasing amount of peptide. As shownin FIG. 4, both peptides can not induce leakage of the vesicle when thepeptide concentration is below 0.05 μM. With increase of peptideconcentration, the leakage percentage increases quickly. For peptide 2,the leakage percentage jumps to 100% within the range between 0.05 μMand 0.14 μM where the corresponding fractions of lipid bound are about10% 40%, respectively. A relatively slower change was observed forpeptide 1. The percentage of leakage increases to about 80% when thepeptide concentration reaches 0.5 μM. The lysis ability stays at aplateau until the peptide concentration is pushed up to 2 μM, when about60% of lipid molecules are bound to peptide molecules. Complete lysis ofthe vesicle at 285 seconds is induced when 5 μM of peptide 1 was added,and the calculated lipid bound fraction is about 80%. The differencebetween the two peptides is even more obvious when looking at theleakage rate right after the addition of peptide to the vesiclesolution, as shown in FIG. 3. The initial leakage rate for peptide 2increases sharply when the peptide concentration increases from 0.14 μMto 0.19 μM. Complete lysis of the vesicle is finished within 20 s withpeptide concentration higher than 0.5 μM. For peptide 1, the leakageinitial rate increases in a continuous fashion, and 100% leakage is notcomplete until 300 s with 5.18 μM peptide.

Numerous studies on natural antibacterial peptides have revealed thatthe distribution of hydrophobic face and polar face on their amphiphilicstructure account for their selectivity. Increased hydropobicity of apeptide can increase its binding affinity to the charge neutral membraneof mammalian cells, thus reducing the selectivity of this peptide. GroupII peptides 1 and 2 are less hydrophobic than the peptides of Group I.This may at least in part result from replacement of residueshomo-Leucine and homo-Valine in the peptides of Group I with lesshydrophobic homo-Alanine. The results from the antibacteria assay andhemolysis experiment indicate that by reducing the hydrophobicity of thepeptide, the selectivity is remarkably enhanced.

The functional mechanism is thought to be different for peptide 1 ascompared to peptide 2. For example, the binding study shows that peptide2 has a higher affinity to SOPS/SOPC vesicles than peptide 1. Inaddition, peptide 2 can bind weakly to charge neutral SOPC, whilepeptide 1 cannot. This suggests that due to its longer size, peptide 2has higher overall hydrophobicity, or its helical structure is morestable. The higher affinity to lipid bilayer of peptide 2 reduces itsability to differentiate bacterial cell from mammalian cell, thuslowering its selectivity.

Two mechanisms have been proposed for the cell killing process byantibacterial B-peptide. In the carpet mechanism, peptides aggregateparallel on the membrane surface, The accumulated peptide molecules wrapthe membrane surface in a carpet-like manner, and causes the membranethinner and eventually produce cavities after the peptide concentrationreaches to a threshold value. The so-called barrel-stave mechanismsuggests that the bound peptides on the cell surface self associate intohelix bundles, and insert into the membrane to form cores on it. Thelatter mechanism is more suitable for hemolytic toxins such as mellitin,whose sequence is more helix stabilizing. It becomes more likely thatthe two mechanisms are two aspects of the real process. From experimentsinvestigating peptide-induced leakage of liposomal content, the twopeptides behave through similar mechanism. When peptide concentration isover 0.05 μM, they cause leakage of the vesicle, and the lysis abilityincreases gradually with increased amounts of peptide. However, whenpeptide concentration is more than 0.1 μM, the lysis ability of peptide2 increases sharply in a cooperating manner, when less than half of thelipid molecules are peptide bound. This suggests a second binding sitefor peptide 2. The second site may come from the self-association ofpeptide 2 after deposit on the membrane. In contrast, the lysis abilityof peptide 1 increases in a more gradual way corresponding toconcentration increase. The carpet mechanism is more suitable to be usedto describe this process. There may be formation of small aggregates ofpeptide 1 when the peptide concentration is high and the first bindingsite is close to saturation. Peptide 2 functions similarly as peptide 1at very low peptide concentration. After the bound fraction of lipidreaches more than about 40%, the peptides very likely oligomerize on themembrane and induce leakage in the barrel-stave fashion. The thresholdmay actually be less than 40%. The CD of peptide 2 in aqueous solventwith high phosphate concentration indicates helix formation, suggestingits tendency to self-associate when the electrostatic repulsion getsdiluted.

It appears as if a length of 14 residues is favorable for selectivity ofthis peptides according to the present invention. Magainindifferentiates bacterial cells from mammalian cells at a level of 300times. Values for the peptides according to the present invention areclose to this value. Additionally, the peptides according to the presentinvention may be altered to step closer to or exceed naturalantibacterial peptides. The changes may be so as to generate peptideswith more diversity in the sequence so as to achieve the best balance.

The foregoing description of the invention illustrates and describes thepresent invention. Additionally, the disclosure shows and describes onlythe preferred embodiments of the invention, but as aforementioned, it isto be understood that the invention is capable of use in various othercombinations, modifications, and environments and is capable of changesor modifications within the scope of the inventive concept as expressedherein, commensurate with the above teachings, and/or the skill orknowledge of the relevant art. The embodiments described hereinabove arefurther intended to explain best modes known of practicing the inventionand to enable others skilled in the art to utilize the invention insuch, or other, embodiments and with the various modifications requiredby the particular applications or uses of the invention. Accordingly,the description is not intended to limit the invention to the formdisclosed herein. Also, it is intended that the appended claims beconstrued to include alternative embodiments.

We claim:
 1. An antibacterial β-peptide having the following formula:

wherein R₁ is H, an alkyl group having 1-4 carbon atoms, phenyl,heteroaryl, or an alkyl-aryl; R₂ is an amine-containing alkyl grouphaving the formula —(CH₂)_(m)NH₂, wherein m=1, 2, 3, 4, or 5,(CH₂)_(x)NHC═NHNH₂ wherein x is 1, 2, 3, 4, or 5, a pyridyl, analkylpryidyl, an amidine-substituted benzyl, a phenyl group, or a cyclicamidine; R₃ is H, an alkyl group having 1-4 carbon atoms, phenyl,heteroaryl, or an alkyl-aryl; X is —NH₂, —OH, —NHR, or OR where R isalkyl, aryl or acyl groups either free or polymer-supported, acarboxamide, a substituted carboxamide, or a polymer; Y is H, an alkylgroup, an acyl group, an acyl-terminated polymer, a sulphonamide, anether, a urea, a urethane, or a polymer; and n is 2, 3, 4, 5, 6, or 7;wherein at least one of X and Y is a polymer selected from the groupconsisting of polyurethane, polyetherurethane, polyester, silicone,polyamide, polyolefin, polystyrene, polypeptide, polysaccharide,cellulosic, and silk.
 2. The β-peptide according to claim 1, wherein atleast one of X and Y further includes at least one α-amino acid attachedto the peptide.
 3. The β-peptide according to claim 1, wherein theβ-peptide is linked to the polymer by a non-cleavable linker.
 4. Theβ-peptide according to claim 1, wherein the peptide can form a L+2helix.
 5. The β-peptide according to claim 1, wherein the alkyl groupsof R₁ and R₃ are methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl,or tert-butyl.
 6. The β-peptide according to claim 1, wherein theβ-peptide is non-covalently bonded to a carrier.
 7. The β-peptideaccording to claim 6, wherein the carrier is a sol-gel or an aero gel.8. An antibacterial β-peptide having the following formula: Y—(T)_(n)—Xwherein n is 2, 3, 4, 5, 6, or 7; X is —NH₂, —OH, —NHR, or OR where R isalkyl, aryl or acyl groups either free or polymer-supported, acarboxamide, a substituted carboxamide, or a polymer; Y is H, an alkylgroup, an acyl group, an acyl-terminated polymer, a sulphonamide, anether, a urea, a urethane, or a polymer; and T is a triplet comprisingT1-T2-T3, wherein T1 comprises a hydrophobic β-amino acid, T2 comprisesa polar or basic amino acid, T3 comprises either a hydrophobic β-aminoacid or a polar or basic β-amino acid, wherein at least one-half of thenumber of triplets includes a basic β-amino acid; wherein at least oneof X and Y is a polymer selected from the group consisting ofpolyurethane, polyetherurethane, polyester, silicone, polyamide,polyolefin, polystyrene, polypeptide, polysaccharide, cellulosic, andsilk.
 9. The β-peptide according to claim 8, wherein at least one of Xand Y further includes at least one α-amino acid or at least one β-aminoacid attached to T.
 10. The β-peptide according to claim 9, wherein theat least one β-amino acid is selected from the group consisting of ahydrophobic, polar, and basic β-amino acid.
 11. The β-peptide accordingto claim 8, wherein the β-amino acids are substituted at at least one ofC2 and C3 with a substituent comprising aryl, or a C1-10 straight orbranched, linear or cyclic, substituted or unsubstituted alkane, alkene,or alkyne, —H, —CH₃, —CH(CH₃)₂, —CH₂—CH(CH₃)₂, —CH(CH₃)CH₂CH₃,—CH₂-phenyl, —CH₂-pOH-phenyl, —CH₂-indole, —CH₂—SH, —CH₂—CH₂—S—CH₃,CH₂OH, —CHOH—CH₃, —CH₂—CH₂—CH₂—CH₂—NH₂, —CH₂—CH₂—CH₂—NH—C(NH)NH₂,—CH₂-imidazole, —CH₂—COOH, —CH₂—CH₂—COOH, —CH₂—CONH₂, —CH₂—CH₂—CONH₂, ortogether with an adjacent —NH group forms a proline amino acid residue,and wherein the stereochemistry of the β-peptide is in an aldolconfiguration.
 12. The β-peptide according to claim 11, wherein at leastone of the substituents is substituted and the substituent on thesubstituent is aryl, or a C1-10 straight or branched, linear or cyclicalkane, alkene, or alkyne, —H, —CH₃, —CH(CH₃)₂, —CH₂—CH(CH₃)₂,—CH(CH₃)CH₂CH₃, —CH₂-phenyl, —CH₂-pOH-phenyl, —CH₂-indole, —CH₂—SH,—CH₂—CH₂—S—CH₃, CH₂OH, —CHOH—CH₃, —CH₂—CH₂—CH₂—CH₂—NH₂,—CH₂—CH₂—CH₂—NH—C(NH)NH₂, —CH₂-imidazole, —CH₂—COOH, —CH₂—CH₂—COOH,—CH₂—CONH₂, —CH₂—CH₂—CONH₂, or together with an adjacent —NH group formsa proline amino acid residue.
 13. The β-peptide according to claim 8,wherein the β-peptide is non-covalently bonded to a carrier.
 14. Theβ-peptide according to claim 13, wherein the carrier is a sol-gel or anaero gel.
 15. The β-peptide according to claim 8, wherein each T is thesame.
 16. The β-peptide according to claim 8, wherein the peptidecomprises a copeptide including repeating groups of triplets whereineach triplet is not the same.
 17. An antibacterial composition,comprising: an antibacterial β-peptide having the following formula

wherein R₁ is H, an alkyl group having 1-4 carbon atoms, phenyl,heteroaryl, or an alkyl-aryl; R₂ is an amine-containing alkyl grouphaving the formula —(CH₂)_(m)NH₂, wherein m=1, 2, 3, 4, or 5,(CH₂)_(x)NHC═NHNH₂ wherein x is 1, 2, 3, 4, or 5, a pyridyl, analkylpryidyl, an amidine-substituted benzyl, a phenyl group, or a cyclicamidine; R₃ is H, an alkyl group having 1-4 carbon atoms, phenyl,heteroaryl, or an alkyl-aryl; X is —NH₂, —OH, —NHR, or OR where R isalkyl aryl or acyl groups either free or polymer-supported, acarboxamide, a substituted carboxamide, or a polymer; Y is H, an alkylgroup, an acyl group, an acyl-terminated polymer, a sulphonamide, anether, a urea, a urethane, or a polymer; and n is 2, 3, 4, 5, 6, or 7;and a carrier, wherein the β-peptide is covalently bonded to thecarrier.
 18. An antibacterial β-peptide having the following formula:

wherein R₁ is H, an alkyl group having 1-4 carbon atoms, phenyl,heteroaryl, or an alkyl-aryl; R₂ is an amine-containing alkyl grouphaving the formula —(CH₂)_(m)NH₂, wherein m=1, 2, 3, 4, or 5,(CH₂)_(x)NHC═NHNH₂ wherein x is 1, 2, 3, 4, or 5, a pyridyl, analkylpryidyl, an amidine-substituted benzyl, a phenyl group, or a cyclicamidine; R₃ is H, an alkyl group having 1-4 carbon atoms, phenyl,heteroaryl, or an alkyl-aryl; X is —NH₂, —OH, —NHR, or OR where R isalkyl, aryl or acyl groups either free or polymer-supported, acarboxamide, a substituted carboxamide, or a polymer; Y is H, an alkylgroup, an acyl group, an acyl-terminated polymer, a sulphonamide, anether, a urea, a urethane, or a polymer; and n is 2, 3, 4, 5, 6, or 7;wherein the β-peptide is non-covalently bonded to a carrier, wherein thecarrier is a sol-gel or an aero gel.
 19. An antibacterial β-peptidehaving the following formula: Y—(T)_(n)—X wherein n is 2, 3, 4, 5, 6, or7; X is —NH₂, —OH, —NHR, or OR where R is alkyl, aryl or acyl groupseither free or polymer-supported, a carboxamide, a substitutedcarboxamide, or a polymer; Y is H, an alkyl group, an acyl group, anacyl-terminated polymer, a sulphonamide, an ether, a urea, a urethane,or a polymer; and T is a triplet comprising T1-T2-T3, wherein T1comprises a hydrophobic β-amino acid, T2 comprises a polar or basicamino acid, T3 comprises either a hydrophobic β-amino acid or a polar orbasic β-amino acid, wherein at least one-half of the number of tripletsincludes a basic β-amino acid; wherein the β-peptide is non-covalentlybonded to a carrier, and wherein the carrier is a sol-gel or an aerogel.
 20. An antibacterial peptide having the following formula:

wherein R₁ is H, an alkyl group having 1-4 carbon atoms, phenyl,heteroaryl, or an alkyl-aryl; R₂ is an amine-containing alkyl grouphaving the formula —(CH₂)_(m)NH₂, wherein m=1, 2, 3, 4, or 5,(CH₂)_(x)NHC═NHNH₂ wherein x is 1, 2, 3, 4, or 5, a pyridyl, analkylpryidyl, an amidine-substituted benzyl, a phenyl group, or a cyclicamidine; R₃ is H, an alkyl group having 1-4 carbon atoms, phenyl,heteroaryl, or an alkyl-aryl; X is —NH₂, —OH, —NHR, or OR where R isalkyl, aryl or acyl groups either free or polymer-supported, acarboxamide, a substituted carboxamide, or a polymer; Y is H, an alkylgroup, an acyl group, an acyl-terminated polymer, a sulphonamide, anether, a urea, a urethane, or a polymer; and n is 2, 3, 4, 5, 6, or 7;and further comprising at least one α-amino acid arranged between atleast one of X and the peptide and at least one of Y and the peptide.